This invention pertains to the general field of microbiology, and more specifically to transfer, inoculation and/or streaking of microorganisms, e.g. for the purpose of obtaining individual colonies. Among others, it relates to methods for handling and streaking microbial specimens and to a (semi-) automated apparatus to prepare specimens for visual counting, typing and other analysis of bacterial populations, for example in diagnostic microbiology.
One of the many procedures, which must be performed in microbiology, is plate streaking with the purpose of isolating microbiological colonies. The isolated colonies are absolutely necessary for the observance of colony morphology and for the performance of staining and other procedures that are necessary for determining the genus and in many cases, the species, strain, etc. of an unknown organism. In the classic technique, known as streaking, a sterile loop is first brought into contact with the sample or inoculum whose cells one wishes to transfer; it is then brought into contact with the plate containing sterile solid medium to which one wishes to transfer the cells, and is moved back and forth over the surface of a portion (e.g. one quarter) of the fresh plate. In the initial step, millions of cells may be transferred to the new plate, and must be further diluted by many orders of magnitude so that individual colonies, rather than confluent growth, will be obtained. Since the loop surface is also now contaminated with many thousands of cells, it must be either resterilized (e.g., by flame sterilization in the case of wire loops), or substituted by a new sterile loop (e.g. in the case of single-use, disposable plastic loops). The sterile loop is then brought into contact with the surface of the fresh plate which has been previously streaked, e.g. by several movements across the zone of the first streaking, and then another portion of the plate (e.g. a quarter of the plate adjacent to the first streaking) is streaked. This results in additional dilution of the cells. The loop is then either resterilized or replaced, is brought into contact with the second streaking area, and then a third zone of the plate is streaked. This process must be performed at least three or four times in order to be certain of obtaining individual colonies, rather than confluent growth, following incubation of the fresh plate.
The testing of clinical microbial specimens typically requires the streaking of several (e.g. 2 to 6) plates per test specimen. The manual streaking process requires approximately 30 seconds per agar plate of a skilled technologist's time. The quality of the streak and therefore the degrees of isolation of the micro-organism depends upon the training received by the technologist and the care taken in performing the process.
Thus, the manual inoculation and streaking process is very labour-intensive and time-consuming. Furthermore, in many diagnostic settings, such as a hospital microbial laboratory, there is often a peak load of samples coming in to be tested. It is not uncommon that the processing of up to a 1000 individual clinical samples of various types must be accomplished in a time window of only a few hours, typically at the end of a normal working day, like between 3 and 5 μm. This puts an enormous time pressure on the employees, equipment and laboratory space involved. The inoculation and streaking is however only the beginning of the actual microbial testing, and peak loads are also encountered for downstream procedures, including plate incubation, processing (e.g. staining) and the final plate assessment.
Therefore, there have been attempts in the field to automate the plate inoculation and/or streaking process.
For example, the ISOPLATER 180 (Vista Technology Inc., Edmonton, Alberta, Canada) is an automated Petri dish streaker. The machine automatically rotates the load carousel to bring in a stack of plates, downloads a dish, removes the lid, orients the dish, transfers the dish with its lid, streaks in four successive quadrants for isolation, replaces the lid and uploads the completed dish into the unload carousel. Spreading over the entire surface of an agar plate is accomplished by four individual nichrome loops which are sterilized by electrical heating between dishes.
WO2005/071055 discloses a streaker device and apparatus for streaking a microbial inoculum for single colonies on the surface of a solid growth medium. The device is characterized by a “comb-like” structure consisting of a row of spaced apart contact surfaces that are resiliently supported by a common support member. The streaking device is applied to the surface of an agar plate and is rotated to various degrees. Prior art dating back to the early eighties discloses streaking systems comprising the use of a spherical, magnetic metal particle, in particular steel balls. For instance, U.S. Pat. No. 3,830,701 describes a method and an apparatus for streaking a microbial sample comprising the contact of a stainless steel ball with an inoculum and generating a controlled motion of the ball using a moving magnet to transfer the microbial sample to a culture surface. U.S. Pat. Nos. 3,660,243 and 3,623,958 disclose methods and devices comprising similar features.
The systems known in the art will automate and replace many of the manual tasks traditionally involved in the inoculation and streaking of standard agar plates. However, they do not accommodate all of the user's preferences. These include: a) production of single colonies when using miniaturized or split plates; b) no cross-contamination; c) ease of use; d) large capacity (preferably at least 500 plates/hour); e) low cost and f) compatibility with different specimen types, e.g. liquid samples, swabs etc. and non-standard plates, such as split-plates.